Autonomous Calibration for Optical Analysis System

ABSTRACT

The present invention provides an autonomous calibration of a multivariate based spectroscopic system that is preferably implemented as a multivariate based spectrometer. The spectroscopic system is based on a multivariate optical element that provides a spectral weighting of an incident optical signal. Spectral weighting is performed on the basis of spatial separation of spectral components and subsequent spatial filtering by means of a spatial light modulator. Calibration of the spectroscopic system is based on a dedicated calibration segment of the spatial light modulator, whose position corresponds to a characteristic calibration or reference wavelength of the incident optical signal. Preferably, the calibration or reference wavelength is given by the wavelength of the excitation radiation generated by the optical source that serves to induce scattering processes in a volume of interest.

The present invention relates to the field of optical spectroscopy.

Spectroscopic techniques are widely used for determination of thecomposition of a substance. By spectrally analyzing an optical signal,i.e. a spectroscopic optical signal, the concentration of a particularcompound of the substance can be precisely determined. The concentrationof a particular substance is typically given by an amplitude of aprincipal component of an optical signal.

In the prior art optical analysis system for determining an amplitude ofa principal component of an optical signal are well known. The knownoptical analysis systems are typically part of a spectroscopic analysissystem suited for, e.g., analyzing which compounds are comprised atwhich concentrations in a sample. It is well known that lightinteracting with the sample carries away information about the compoundsand their concentrations. The underlying physical processes areexploited in optical spectroscopic techniques in which light of a lightsource such as, e.g., a laser, a lamp or light emitting diode isdirected to the sample for generating an optical signal which carriesthis information.

For example, light may be absorbed by the sample. Alternatively or inaddition, light of a known wavelength may interact with the sample andthereby generate light at a different wavelength due to, e.g. a Ramanprocess. The transmitted and/or generated light then constitutes theoptical signal, which may also be referred to as the spectrum. Therelative intensity of the optical signal as function of the wavelengthis then indicative for the compounds comprised in the sample and theirconcentrations.

To identify the compounds comprised in the sample and to determine theirconcentrations the optical signal has to be analyzed. In the knownoptical analysis system the optical signal is analyzed by dedicatedhardware comprising an optical filter. This optical filter has atransmission which depends on the wavelength, i.e. it is designed toweight the optical signal by a spectral weighting function which isgiven by the wavelength dependent transmission. The spectral weightingfunction is chosen such that the total intensity of the weighted opticalsignal, i.e. of the light transmitted by the filter, is directlyproportional to the concentration of a particular compound. Such anoptical filter is also denoted as multivariate optical element (MOE).This intensity can then be conveniently detected by a detector such as,e.g., a photodiode. For every compound a dedicated optical filter with acharacteristic spectral weighting function is used. The optical filtermay be, e.g., an interference filter having a transmission constitutingthe desired weighting function.

For a successful implementation of this analysis scheme it is essentialto know the spectral weighting functions. They may be obtained, e.g., byperforming a principal component analysis of a set comprising N spectraof N pure compounds of known concentration where N is an integer. Eachspectrum comprises the intensity of the corresponding optical signal atM different wavelengths where M is an integer as well. Typically, M ismuch larger than N. Each spectrum containing M intensities atcorresponding M wavelengths constitutes an M dimensional vector whose Mcomponents are these intensities. These vectors are subjected to alinear-algebraic process known as singular value decomposition (SVD)which is at the heart of principal component analysis and which is wellunderstood in this art.

As a result of the SVD a set of N eigenvectors z_(n) with n being apositive integer smaller than N+1 is obtained. The eigenvectors z_(n)are linear combinations of the original N spectra and often referred toas principal components or principal component vectors. Typically, theprincipal components are mutually orthogonal and determined asnormalized vectors with |z_(n)|=1. Using the principal components z_(n),the optical signal of a sample comprising the compounds of unknownconcentration may be described by the combination of the normalizedprincipal components multiplied by the appropriate scalar multipliers:

x ₁ z ₁ +x ₂ z ₂ + . . . +x _(n) z _(n),

The scalar multipliers x_(n) with n being a positive integer smallerthan N+1 may be considered the amplitudes of the principal componentsz_(n) in a given optical signal. Each multiplier x_(n) can be determinedby treating the optical signal as a vector in the M dimensionalwavelength space and calculating the direct product of this vector witha principal component vector z_(n).

The result yields the amplitude x_(n) of the optical signal in thedirection of the normalized eigenvector z_(n). The amplitudes x_(n)correspond to the concentrations of the N compounds.

In known optical analysis systems the calculation of the direct productbetween the vector representing the optical signal and the eigenvectorrepresenting the principal component is implemented in the hardware ofthe optical analysis system by means of the optical filter. The opticalfilter has a transmittance such that it weighs the optical signalaccording to the components of the eigenvector representing theprincipal component, i.e. the principal component vector constitutes thespectral weighting function. The filtered optical signal can be detectedby a detector which generates a signal with an amplitude proportional tothe amplitude of the principal component and thus to the concentrationof the corresponding compound.

Especially, when the optical analysis system is dedicated to determinethe concentration of a single compound of a substance, like e.g. glucoseconcentration in blood, it is advantageous to make use of acorresponding optical filter, that is designed for the spectralweighting function of this particular compound. Such dedicated opticalfilters can be realized in a cost efficient way because they do not haveto provide reconfigurable transmission or absorption properties. Opticalanalysis systems dedicated for determination of the concentration of aparticular compound may therefore be implemented on the basis of alow-cost MOE, that can be implemented on the basis of a dispersiveoptical element, such as a prism or a grating and a correspondingoptical filter providing a spatial transmission pattern.

Here, an optical signal received from a sample carrying spectralcomponents being indicative of the composition of the sample is incidenton the dispersive optical element. By means of the dispersive opticalelement, the incoming optical signal is spatially decomposed intovarious spectral components. Hence, the dispersive optical elementserves to spatially separate the spectral components of the incidentoptical signal. Preferably, the evolving spectrum spreads along adirection specified by the dispersive optical element. For example, thespectrum might spread along a first direction, e.g. horizontally.

Making use of a dedicated spatial transmission mask inserted into theoptical path of the spectrum, dedicated spectral components of theevolving spectrum can be attenuated or even entirely blocked. Therefore,the spatial transmission mask has to provide a plurality of areasfeaturing different transmission properties. When the spectrum is spreadin a horizontal direction, these areas of the spatial transmission maskhave to be aligned horizontally, thereby providing a uniformtransmission in the vertical direction.

Additionally, by uniformly expanding the spectrum in a verticaldirection, the spatial transmission mask might be divided in two, ormore, sections being aligned in a vertical direction. Each section maythen feature different spatial transmission patterns allowing tosimultaneously manipulate the spectrum in two, or more, different ways.

Consequently, when vertically divided in two sections, the upper sectionof the spatial transmission mask may effectively serve as a firstspectral weighting function whereas the lower section of the spatialtransmission mask may provide a second spectral weighting function. Byseparately detecting these two differently manipulated spectra, positiveand negative parts of a principal component can be separately detected,thus allowing for an effective and sufficient amount of information inorder to determine an amplitude corresponding to the concentration ofthe dedicated compound. For example, by mutually subtracting positiveand negative part of the spectral weighting function, a signal beingindicative of the compounds' concentration might be precisely derived.

Usage of dedicated spatial transmission masks in combination withdispersive optical elements effectively provides a low-costimplementation of an optical analysis system. However, because thespectral components of the received optical signal are spatially spread,the spatial transmission mask has to be properly aligned in order toprovide accurate spectral attenuation of dedicated spectral componentsof the optical signal. The relative positioning of an evolving spectrumand the spatial transmission mask is rather critical and a slightdisplacement of either the spectrum or the transmission mask mayseriously affect the result of the optical analysis. Therefore, anaccurate and reliable calibration mechanism is required forspectroscopic analysis that is based on multivariate optical analysis ofan optical signal.

The present invention aims to provide calibration of an optical analysissystem without implementation of a light source that is dedicated forcalibration. In contrast the invention aims to provide calibration onthe basis of a spectroscopic optical signal.

The present invention provides a spectroscopic system for determining aprincipal component of an optical signal comprising return radiationfrom a volume of interest. The spectroscopic system comprises a lightsource for generating an excitation radiation. The excitation radiationis adapted to be transmitted into the volume of interest. Thespectroscopic system further comprises an objective for collectingreturn radiation from the volume of interest. It therefore serves tocollect the optical signal that returns as e.g. scattered radiation fromthe volume of interest. The spectroscopic system further comprises adispersive optical element for spatially separating the spectralcomponents of the return radiation in a first direction and spatiallight manipulation means for modulating the spectral components of thereturn radiation. The spatial light manipulation means further have areference segment at a first position. This reference segment is atleast partially transparent for the excitation radiation, i.e. thereference segment is at least partially transparent for radiation thathas substantially the same wavelength as the excitation radiation thatis transmitted into the volume of interest. The spectroscopic systemfurther comprises at least a first detector for detecting radiation thatis transmitted through the reference segment of the spatial lightmanipulation means. Finally, the spectroscopic system further comprisesa control unit that is adapted to calibrate the spectroscopic system onthe basis of radiation that is detected by means of the at least firstdetector.

The dispersive optical element in combination with the spatial lightmanipulation means represent a multivariate optical element (MOE).Typically, the spatial light manipulation means comprise a spatialtransmission pattern for selectively blocking or attenuating variousspectral components of the optical signal. In this way a spectralweighting function can be effectively realized that in turn allows todetermine the concentration of a dedicated compound inside the volume ofinterest. The position of the spatial light manipulation means withrespect to a dispersed spectrum of the optical signal provided by thedispersive optical element is rather critical and has a severe impact onthe spectral filtering provided by the MOE.

Additionally, not only the relative position of a spatially dispersedspectrum and a spatial transmission mask but also the light emissioncharacteristics of the light source that may depend on temperature mayhave a critical impact on the calibration of the optical analysissystem.

The reference segment of the spatial light manipulation means serves asan effective means for calibrating the optical analysis system.Preferably, a calibration is performed on the basis of radiation that istransmitted through the reference segment of the spatial lightmanipulation means and that is detected by means of the at least firstdetector. In particular, the magnitude of the light intensity beingdetected by means of the at least first detector gives a sufficientindication whether the spectroscopic system is accurately calibrated. Incase that the magnitude of detected light intensity indicates aninaccurate calibration, the control unit serves to calibrate thespectroscopic system in a plurality of different ways until the lightintensity that is transmitted through the reference segment of thespatial light manipulation means indicates accurate calibration, i.e.accurate alignment of the optical paths of the optical analysis system.

According to a preferred embodiment of the invention, the referencesegment comprises a slit aperture and the first position substantiallycorresponds to the wavelength of the excitation radiation. Hence, thereference segment is implemented as a slit aperture at a position on thespatial light manipulation means that corresponds to the wavelength ofthe excitation radiation. In this way the excitation radiation serves asa reference for calibration of the optical analysis system. Typically,the excitation radiation features a narrow spectral band that issuitable to induce various scattering processes when focused into thevolume of interest. For example, the wavelength of the excitationradiation may be in the infrared (IR) or in the near infrared (NIR)spectral range. When focused into the volume of interest, numerousscattering processes either of elastic or inelastic type may occur.

The return radiation emanating from the volume of interest may thereforecomprise inelastically as well as elastically scattered components. Forexample, back-scattered inelastic components of the return radiation mayhave been subject to Raman scattering processes whereas elasticallyscattered components of the return radiation may stem from Rayleighscattering leaving the wavelength of the back-scattered componentssubstantially unaffected with respect to the incident excitationradiation. In typical spectroscopic scenarios only a minor portion ofthe return radiation has become subject to an inelastic scatteringprocess, such like a Stokes or Anti-Stokes scattering process.Therefore, only a minor portion of the return radiation is frequencyshifted with respect to the excitation radiation. The spectrum of thereturn radiation therefore inevitably features a peak at the wavelengthof the excitation radiation.

The invention effectively exploits the existence of the excitationradiation's peak in the spectrum of the return radiation. The inherentexistence of this excitation peak therefore effectively provides areference line in the spectrum.

By detecting an intensity of light being transmitted through thereference segment of the spatial light manipulation means and comparingthe detected light intensity with a corresponding intensity that hasbeen detected for an accurately calibrated optical analysis system,reliable information about accurate or inaccurate calibration of theoptical analysis system can be obtained. Alternatively, when a referenceintensity value of an accurately calibrated spectroscopic system is notavailable, the control unit may calibrate the spectroscopic system in aplurality of different ways until the light intensity that istransmitted through the reference segment of the spatial lightmanipulation means reaches, for instance, a maximum value.

In this way the excitation radiation that is inherently present in thespectrum of the optical signal can be effectively exploited forcalibration of the optical analysis system. Hence, no external or noadditional calibration light source has to be implemented into theoptical analysis system. By effectively replacing such a calibrationlight source by means of the excitation radiation, a potentialmisalignment of a calibration light source leading to a dramaticfalsification of the results of the spectroscopic system isintrinsically prevented.

According to a further preferred embodiment of the invention, thecontrol unit is adapted to translate the spatial light manipulationmeans along the first direction. In this way a misalignment of thespatial light manipulation means that may be either implemented as afixed spatial light transmission pattern or a reconfigurable spatiallight modulator (SLM), can be effectively compensated. Preferably, thespatial light manipulation means are mounted on a servo driventranslation stage that can be controlled by means of the control unit.

According to a further preferred embodiment of the invention, thecontrol unit is adapted to control the light source in order to modifythe wavelength of the excitation radiation. In this way the control unitmay tune the wavelength of the light source that generates theexcitation radiation. Typically, this light source is implemented as alaser light source emitting in the NIR range. Due to varying externalconditions, e.g. varying temperature, the wavelength of the emittedexcitation radiation might become subject to a remarkable drift oroffset. This may have dramatic consequences for the scattering processesinduced by the excitation radiation inside the volume of interest. Insuch a case, the spectrum of the return radiation might be shiftedcorrespondingly and/or the efficiency of the spectroscopic process maydecrease.

Additionally, the intensity being transmitted through the referencesegment of the spatial light manipulation means may no longer correspondto the expected maximum intensity that can be measured when thespectroscopic system is correctly calibrated. Hence, in response to adetection of a decrease of intensity being transmitted through thereference segment, the control unit may stepwise modify the calibrationof a laser light source in order to compensate, for instance, atemperature based offset of the excitation light source.

According to a further preferred embodiment of the invention, thecontrol unit is further adapted to rotate or to translate the dispersiveoptical element. In this way a position mismatch of the spectrumevolving from the dispersive optical element and the spatial lightmanipulation means can be compensated. Preferably, the dispersiveoptical element is mechanically coupled to a rotation and/or translationstage that can be electrically-controlled by means of the control unit.

According to a further preferred embodiment of the invention, thespatial light manipulation means are implemented as modifiable spatiallight manipulation means. In this embodiment the control unit is furtheradapted to modify the spatial light manipulation means, i.e. to modifythe spatial light transmission pattern of the spatial light manipulationmeans. For example the spatial light manipulation means can beimplemented on the basis of a liquid crystal (LC-cell) in combinationwith an arrangement of crossed polarizers.

The LC-cell is preferably electrically controllable and providesmanipulation of the spatially dispersed spectrum. In this embodimentcalibration of the spectroscopic system can be realized by amodification of the spatial light manipulation means. Instead ofshifting the spatial light manipulation means itself, here, the spatiallight transmission pattern of the spatial light modulator can be shiftedalong the first direction, i.e. in the direction of the spatialseparation of the spectral components of the return radiation by keepingthe position of the spatial light modulator constant. Implementing thespatial light manipulation means as spatial light modulator alsorequires realizing the reference segment in a reconfigurable way. Hence,reconfiguration of the spatial light modulator equally refers toreconfiguration of the spatial light transmission mask as well as to amodification of the position of the reference segment.

Consequently, the spectroscopic system can be calibrated by means of thecontrol unit in a plurality of different ways, either by translating thespatial light manipulation means, by modifying or reconfiguring thespatial light manipulation means, by rotating or translating thedispersive optical element or by calibrating or tuning the light sourcethat generates the excitation radiation. Calibration of thespectroscopic system may be performed on the basis of a single one or onthe basis of several ones of the above described calibration techniques.For example, calibration of the spectroscopic system can be performed bytuning the laser light source in combination with a translation of thespatial light manipulation means. The various calibration techniques mayeither be performed simultaneously or sequentially or in any otherarbitrary order.

According to a further preferred embodiment of the invention, the atleast first detector further comprises a segmented detector that has atleast two separate detector segments that are separated along the firstdirection. like e.g. a split detector. In this way a misalignment of thespatial light manipulation means can be directly classified. Whenever adetector segment detects a transmitted intensity that is larger than theintensity detected by the other detector segment, this gives a clearindication that the spatial light manipulation means are improperlypositioned within the optical analysis system. Depending on thearrangement of the two detector segments, also the direction of thespatial light manipulation means' misplacement can be determined. Thisallows for a faster calibration of the optical analysis system.

According to a further preferred embodiment of the invention, the atleast first detector is directly integrated into the spatial lightmanipulation means at the first position. The at least first detector iseither rigidly attached behind the slit aperture of the spatial lightmanipulation means or when available in an appropriate size, thedetector itself may represent the slit aperture. In both cases nofurther optical components are required to transmit or to focus thelight that has been transmitted through the reference segment onto theat least first detector.

In another aspect the invention provides a method of calibrating aspectroscopic system that has a light source for generating anexcitation radiation and an objective for collecting return radiationfrom a volume of interest. The return radiation is generated on thebasis of scattering processes induced by the excitation radiation insidethe volume of interest. The spectroscopic system further has adispersive optical element for spatially separating spectral componentsof the return radiation in a first direction and spatial lightmanipulation means for modulating the spectral components of the returnradiation. The method of calibrating of the spectroscopic systemcomprises detecting radiation that is transmitted through a referencesegment of the light manipulation means by means of an at least firstdetector. The reference segment is at least partially transparent forthe excitation radiation and is located at a first position on thespatial light manipulation means. The method of calibrating furthercomprises calibrating the spectroscopic system on the basis of radiationbeing detected by means of the at least first detector.

According to a preferred embodiment, calibrating of the spectroscopicsystem comprises maximizing of radiation being transmitted through thereference segment. The reference segment and in particular the firstposition specifying the lateral position of the reference segment on thespatial light manipulation means corresponds to the position of thespectral line of the excitation radiation. Hence, the wavelength of theexcitation radiation serves as a reference for the calibration of theoptical analysis system. Since the return radiation and hence theoptical signal that is subject to spectroscopic analysis intrinsicallyhas a spectral component that corresponds to the wavelength of theexcitation radiation, no additional light source for calibrating thespectroscopic system is required. In this way erroneous calibration thatis due to insufficient positioning or insufficient alignment of thecalibrating light source is not present.

According to a further preferred embodiment of the invention,calibrating of the spectroscopic system either comprises translating thespatial light manipulation means along the first direction and/ormodifying the wavelength of the excitation radiation by means of thecontrolling the light source, and/or rotating and/or translating thedispersive optical element and/or reconfiguring a spatial transmissionpattern of the spatial light manipulation means.

In still another aspect, the invention provides a computer programproduct for calibrating an optical analysis system. The spectroscopicsystem has a light source for generating an excitation radiation andmeans for transmitting the excitation radiation into the volume ofinterest. The spectroscopic system further has an objective forcollecting return radiation from the volume of interest, a dispersiveoptical element for spatially separating spectral components of thereturn radiation in a first direction and spatial light manipulationmeans for modulating the spectral components of the return radiation.The computer program product comprises computer program means that areadapted to store a first output signal of an at least first detector.The first output signal is generated in response to detect a radiationbeing transmitted through a reference segment of the spatial lightmanipulation means.

The computer program means are further adapted to store a second outputsignal of the at least first detector after a modification of theposition of the spatial light manipulation means along the firstdirection and/or after a modification of the wavelength of theexcitation radiation and/or after a modification of the orientationand/or position of the dispersive optical element and/or after areconfiguration of a spatial transmission pattern of the spatial lightmanipulation means.

The computer program means are further adapted to compare the first andsecond output signal of the at least first detector and to repeatedlycalibrate the spectroscopic system until the output signal of thedetector is indicative of, for instance, a maximum of transmittedradiation.

Further it is to be noted, that any reference signs in the claims arenot to be construed as limiting the scope of the present invention.

In the following preferred embodiments of the invention will bedescribed in detail by making reference to the drawings in which:

FIG. 1 is a schematic diagram of an embodiment of a blood analysissystem,

FIGS. 2 a and 2 b are spectra of the optical signal generated from bloodin the skin and from a sample comprising one anlayte in a solution,

FIG. 3 is a spectral weighting function implemented in a multivariateoptical element,

FIG. 4 schematically illustrates a block diagram of the optical analysissystem,

FIG. 5 schematically illustrates a block diagram of possible detectorconfigurations,

FIG. 6 shows a perspective illustration of the spatial light modulatingmask and a corresponding detector,

FIG. 7 shows a flowchart of calibrating the optical analysis system.

In the embodiment shown in FIG. 1 the spectroscopic system isschematically illustrated. The spectroscopic system has an opticalanalysis system 20 for determining an amplitude of a principal componentof an optical signal. The spectroscopic system further has a lightsource 1 for providing light for illuminating a sample 2 comprising asubstance having a concentration and thereby generating the principalcomponent. The amplitude of the principal component relates to theconcentration of the substance. The light source 1 is a laser such as agas laser, a dye laser and/or a solid state laser such as asemiconductor or diode laser. The optical analysis system 20 is part ofa blood analysis system 22. The blood analysis system further comprisesa computational element 19 for determining the amplitude of theprincipal component, hence for determining the composition of thecompound. The sample 2 comprises skin with blood vessels. The substancemay be one or more of the following analyses: glucose, lactate,cholesterol, oxy-hemoglobin and/or desoxy-hemoglobin, glycohemoglobin(HbAlc), hematocrit, cholesterol (total, HDL, LDL), triglycerides, urea,albumin, creatinin, oxygenation, pH, bicarbonate and many others. Theconcentrations of these substances is to be determined in a non-invasiveway using optical spectroscopy. To this end the light provided by thelight source 1 is sent to a beam splitter 3 which reflects the lightprovided by the light source towards the blood vessels in the skin. Thelight may be focused on the blood vessel using an objective 12. Thelight may be focused in the blood vessel by using an imaging andanalysis system as described in the international patent application WO02/057759.

By interaction of the light provided by the light source 1 with theblood in the blood vessel an optical signal is generated due to Ramanscattering and fluorescence. The optical signal thus generated may becollected by the objective 12 and sent to the dichroic mirror 3. Theoptical signal has a different wavelength than the light provided by thelight source 1. The dichroic mirror is constructed such that ittransmits at least a portion of the optical signal.

A spectrum 100 of the optical signal generated in this way is shown inFIG. 2A. The spectrum comprises a relatively broad fluorescencebackground (FBG) 102 and relatively narrow Raman bands (RB) 104, 106,108. The x-axis of FIG. 2A denotes the wavelength shift with respect tothe 785 nm of the excitation by light source 1 in wave numbers, they-axis of FIG. 2A denotes the intensity in arbitrary units. The x-axiscorresponds to zero intensity. The wavelength and the intensity of theRaman bands, i.e. the position and the height, is indicative for thetype of analyte as is shown in the example of FIG. 2B for the analyteglucose which was dissolved in a concentration of 80 mMol in water. Thesolid line 110 of FIG. 2B shows the spectrum of both glucose and water,the dashed line 112 of FIG. 2B shows the difference between the spectrumof glucose in water and the spectrum of water without glucose. Theamplitude of the spectrum with these bands is indicative for theconcentration of the analyte.

Because blood comprises many compounds each having a certain spectrumwhich may be as complex as that of FIG. 2B, the analysis of the spectrumof the optical signal is relatively complicated. The optical signal issent to the spectroscopic system 20 according to the invention where theoptical signal is analyzed by a MOE which weighs the optical signal by aweighting function shown e.g. schematically in FIG. 3. The weightingfunction of FIG. 3 is designed for glucose in blood. It comprises apositive part P and a negative part N. The positive part P and thenegative part N each comprise in this example more than one spectralband.

Here and in the remainder of this application the distance between afocusing member and another optical element is defined as the distancealong the optical axis between the main plane of the focusing member andthe main plane of the other optical element.

A computational element 19 shown in FIG. 1 is arranged to calculate thedifference between the positive and negative signal. This difference isproportional to the amplitude of the principal component of the opticalsignal. The amplitude of the principal component relates to theconcentration of the substance, i.e. of the analyte. The relationbetween the amplitude and the concentration may be a linear dependence.

FIG. 4 schematically illustrates a block diagram of the blood analysissystem 22 and the optical analysis system 20 in a more detailed way.Here, the optical analysis system 20 is shown as an integral part of theblood analysis system 22. However, the invention is by no meansrestricted to blood analysis but may be universally applied to variousspectroscopic operational areas. In the illustrated embodiment, theoptical analysis system 20 has a dispersive optical element 30, a lens32, a spatial light transmission mask 34 and two detectors 40, 42. Theoutput of the two detectors 40, 42 is coupled into a control unit 60that is in turn adapted to manipulate the path of the optical beamsinside the optical analysis system 20 as well as to calibrate or to tunethe light source 1.

The light source 1 is preferably implemented as a laser light sourceoperating in the near infrared spectral range. The light source producesan optical beam of excitation radiation 50 that is directed towards thebeam splitter 3. The arrangement of beam splitter 3 and objective lens12 serves to focus the excitation radiation 50 into the volume ofinterest 4 of the biological sample 2. Inside the volume of interest theexcitation radiation typically induces a variety of scatteringprocesses. In particular inelastic scattering processes, like Stokes orAnti-Stokes scattering processes lead to a Raman spectrum that allowsfor spectral analysis for determination of the composition of the volumeof interest 4. Typically, a part of the Raman spectrum is back-scatteredand re-enters the objective lens 12 in a counter propagating way withrespect to the excitation radiation 50. The collected return radiationis then transmitted through the beam splitter 3 and becomes incident onthe dispersive optical element 30.

The dispersive optical element 30 can be implemented by e.g. atransmission or reflection grating, a prism or any other dispersiveelement that provides spatial separation of spectral components of anincident light beam. The dispersive element 30 provides spatialseparation of the spectral components of the incident return radiationin the horizontal direction, as shown in FIG. 4. The lens 32 providesfocusing of the various spectral components 52, 54 of the returnradiation to different positions on the spatial light transmission mask34. Depending on the light transmission pattern of the spatial lighttransmission mask 34, the various spectral components of the returnradiation are selective attenuated or even blocked. In this way aspectral weighting function as required for multivariate opticalanalysis can be effectively realized.

For an accurate and reliable determination of a concentration of aparticular compound of the volume of interest 4, the relative positionbetween the evolving spectrum and the spatial light transmission mask 34is of high relevance. Already a slight mismatch between the position ofthe spectrum and the horizontal alignment of the transmission mask 34may lead to an insufficient result of the optical and spectral analysisof the return radiation.

The transmission mask 34 comprises a slit 36 at a first position thatserves as a reference segment. When properly aligned the position of thereference segment 36 corresponds to the position of the spectralcomponent 52 of the return radiation. This spectral component 52 servesas a reference line in the spectrum. Preferably, the reference line isdetermined by the wavelength of the excitation radiation 50. Typically,the spectrum not only comprises spectral components from inelastic butalso from elastic scattering processes that leave the wavelength of theoptical radiation substantially unaffected during a scattering process.Moreover, elastically scattered radiation may even represent a majorportion of the return radiation. The slit 36 is therefore highlytransmissive for the wavelength of the excitation radiation. Hence,radiation that is transmitted through the slit 36 is detected by meansof the detector 40 that is adapted to generate a calibration signal thatis fed into the control unit 60.

Slit 38 of the transmission mask 34 is located at a different horizontalposition compared to slit 36. It therefore serves to attenuate and/or totransmit a spectral component of the return radiation featuring adifferent wavelength than the excitation radiation 50. Typically, thetransmission mask 34 comprises a plurality of slits 38 that are locatedat different horizontal locations on the transmission mask 34. The slits36, 38 might be implemented as slit apertures. Additionally, they mightbe combined with neutral density filters for selectively attenuatingdifferent spectral components of the return radiation to variousdegrees.

The transmission mask 34 can be implemented as a fixed transmission maskproviding a single spatial transmission pattern. In such a case theoptical analysis is dedicated for determining the concentration of asingle particular compound in the volume of interest 4. Variouscompounds or analytes in the volume of interest 4 might be investigatedand spectrally analyzed by replacing the transmission mask 34 by anothercompound specific transmission mask that is dedicated for multivariateoptical analysis of a different compound.

Alternatively, the transmission mask 34 might be implemented as areconfigurable transmission mask. This can for example be realized byimplementing the transmission mask 34 on the basis of a liquid crystalcell in combination with a crossed polarizer arrangement.

Detector 42 serves to detect radiation that has been transmitted throughthe transmission mask 34. Preferably, some kind of focusing means isplaced between transmission mask 34 and detector 42 in order to collecta plurality of transmitted spectral components onto the detector 42.Hence, detector 42 generates an output signal in response to detect aplurality of transmitted spectral components. The output signal ofdetector 42 is therefore directly indicative of the concentration of aparticular compound that can be calculated by means of the computationalelement 19.

The calibration signal generated by means of detector 40 is provided tothe control unit 60 that in turn is adapted to manipulate the horizontalposition of the transmission mask 34, to shift or to rotate thedispersive optical element 30 or to tune the light source 1. Wheneverthe light intensity detected by detector 40 does not correspond to anintensity that is expected for an accurate calibration of the opticalanalysis system, the control unit may successively modify the positionor orientation of the optical components 30, 34 or may tune the lightsource 1 until the intensity detected by detector 40 corresponds to anexpected value. Additionally, the control unit 60 may perform a positionscan of transmission mask 34 in order to maximize the light intensitydetected by detector 40.

In this way the intrinsically present excitation radiation component ofthe collected return radiation can be effectively exploited forcalibration of the optical analysis system 20.

FIG. 5 shows an alternative arrangement of the two detectors 40, 42 withrespect to the spatial light transmission mask 34. Here, detector 40that is adapted for detection of the calibration signal 52 is positioneddirectly behind the slit 36 of the transmission mask 34. In this way noadditional optical means, like lenses are required for directing thetransmitted radiation onto the detector 40. Additionally, bymechanically fixing or mechanically coupling detector 40 to thetransmission mask 34, the detector 40 follows any movement of thetransmission mask 34.

Moreover, FIG. 5 also depicts a collection lens 56 that serves to focustransmitted spectral components of the return radiation 54 onto thedetector 42 for multivariate optical analysis.

FIG. 6 shows a perspective illustration of the transmission mask 34 andthe detector 40. The transmission mask 34 features three slit apertures36, 38, 39 that serve to transmit a corresponding spectral component ofthe return radiation. In particular, slit aperture 36 serves asreference segment of the transmission mask 34 and is thereforehorizontally positioned in such a way that it provides transmission ofan excitation radiation spectral component of the return radiation 52.

Detector 40 is implemented as a split detector that has at least twodetection segments 62, 64. In this way the detector 40 not only allowsto determine whether the optical analysis system is not properlycalibrated but also provides information of a type of positionalmismatch between the spatial distribution of the spectrum and thetransmission mask 34. For example, when the two detection segments 62,64 of the detector 40 detect equal transmitted intensity, this gives aclear indication that the optical analysis system is accuratelycalibrated. Whenever one of the two detection segments 62, 64 detects alarger intensity than the other one, information whether thetransmission mask 34 has to be shifted to the left or to the right isdirectly obtained. In this way a calibration of the optical analysissystem can be performed in a less time intensive way, i.e. a scan for amaximum of transmitted intensity has in principle only to be performedalong one direction.

FIG. 7 illustrates a flowchart for performing the inventive calibrationmethod making use of e.g. a photodiode as detector 40. In a first step700 radiation that is transmitted through the reference segment 36 isdetected. This detected light intensity is stored in a subsequent step702 as intensity I_(x). When no reference intensity is stored in thecontrol unit that allows to determine whether the measured intensityI_(x) corresponds to the maximum intensity, thereby indicating that theoptical analysis system is accurately calibrated, in a subsequent step704 a system parameter of the optical analysis system is modified.Modification of a system parameter typically refers to translating ofthe spatial transmission mask 34, translating or rotating of thedispersive optical element 30, tuning or calibrating of the laser lightsource 1 and reconfiguring of the spatial light transmission pattern ofthe transmission mask 34.

Modification of these system parameters either corresponds to asuccessive modification of a single system parameter or to simultaneousor combined modification of a variety of system parameters. After orduring modification of those system parameters performed in step 704radiation that is transmitted through the reference segment isrepeatedly detected in step 706. A corresponding intensity referred toas I_(x+1) is stored in step 708. Thereafter, the detected lightintensities I_(x) and I_(x+1) are compared in step 710. Comparison ofthe two light intensities that correspond to slightly differentcalibration configurations of the optical analysis system typicallyrefers to a comparison of their absolute value.

In case that the successively detected intensity of I_(x+1) is largerthan the previously detected intensity I_(x) the method continues withstep 714 where intensity I_(x) is replaced by the recently detectedintensity I_(x+1). In this way the intensity I_(x) principally refers toa temporary maximum detected intensity. After step 714 the methodreturns to step 704 where again a system parameter of the opticalanalysis system is modified.

In the other case, where the recently detected intensity I_(x+1) is notlarger than the previously detected intensity I_(x), after step 710 themethod continues with step 712, where the last modification performed instep 704 is undone. In this case the modification of a system parameterperformed in 704 did not lead to an improvement of the optical analysissystem's calibration. Therefore, the performed modification iscancelled. After this canceling performed in step 712 the method returnsto step 704, where another system parameter is preferably subject tomodification.

In this way the optical analysis system is iteratively and constantlycalibrated by means of seeking for a maximum of transmitted intensity ofthe reference spectral component.

Consequently, the invention provides an autonomous calibration of amultivariate based spectrometer. By making use of the excitationradiation as calibration or reference line in the obtained spectrum, noadditional light source for calibration of the optical analysis systemis needed.

LIST OF REFERENCE NUMERALS

-   1 light source-   2 sample-   3 beam splitter-   4 volume of interest-   12 objective-   19 computational element-   20 optical analysis system-   22 blood analysis system-   30 dispersive element-   32 lens-   34 spatial light transmission mask-   36 slit-   38 slit-   40 detector-   42 detector-   50 excitation radiation-   52 return radiation-   54 return radiation-   56 lens-   60 control unit-   62 detection segment-   64 detection segment

1. A spectroscopic system for determining a principal component of anoptical signal, the optical signal comprising return radiation from avolume of interest (4), the spectroscopic system comprising: lightsource (1) for generating an excitation radiation, the excitationradiation (50) being adapted to be transmitted into the volume ofinterest, an objective (12) for collecting return radiation from thevolume of interest, a dispersive optical element (30) for spatiallyseparating spectral components of the return radiation in a firstdirection, spatial light manipulation means (34) for modulating thespectral components of the return radiation, the spatial lightmanipulation means further having a reference segment (36) at a firstposition, the reference segment being at least partially transparent forthe excitation radiation, at least a first detector (40) for detectingradiation being transmitted through the reference segment, a controlunit (60) being adapted to calibrate the optical analysis system on thebasis of the radiation being detected by means of the at least firstdetector.
 2. The spectroscopic system according to claim 1, wherein thereference segment (36) comprises a slit aperture and the first positionsubstantially corresponds to the wavelength of the excitation radiation.3. The spectroscopic system according to claim 1, wherein the controlunit (60) is adapted to translate the spatial light manipulation means(34) along the first direction.
 4. The spectroscopic system according toclaim 1, wherein the control unit (60) is adapted to control the lightsource (1) in order to modify the wavelength of the excitation radiation(50).
 5. The spectroscopic system according to claim 1, wherein thecontrol unit (60) is adapted to rotate or to translate the dispersiveoptical element (30).
 6. The spectroscopic system according to claim 1,wherein the spatial light manipulation means (34) are modifiable, thecontrol unit (60) being further adapted to modify the spatial lightmanipulation means.
 7. The spectroscopic system according to claim 1,wherein the at least first detector (40) comprises a segmented detectorhaving at least two detector segments (62, 64) being separated along thefirst direction.
 8. The spectroscopic system according to claim 1,wherein the at least first detector (40) being integrated into thespatial light manipulation means (34) at the first position.
 9. A methodof calibrating a spectroscopic system having a light source (1) forgenerating an excitation radiation (50) being adapted to be transmittedinto a volume of interest (4), an objective (12) for collecting returnradiation from the volume of interest, a dispersive optical element (30)for spatially separating spectral components of the return radiation ina first direction and spatial light manipulation means (34) formodulating the spectral components of the return radiation, the methodof calibrating comprising the steps of: detecting radiation beingtransmitted through a reference segment (36) of the spatial lightmanipulation means by means of an at least first detector (40), thereference segment being at least partially transparent for theexcitation radiation and being located at a first position on thespatial light manipulation means, calibrating the spectroscopic systemon the basis of radiation being detected by means of the at least firstlight detector.
 10. The method according to claim 9, wherein calibratingof the spectroscopic system comprising maximizing the radiation beingtransmitted through the reference segment.
 11. The method according toclaim 9, wherein calibrating of the spectroscopic system furthercomprising the steps of: translating the spatial light manipulationmeans along the first direction, and/or modifying the wavelength of theexcitation radiation by means of controlling the light source, and/orrotating and/or translating the dispersive optical element, and/orreconfiguring a spatial transmission pattern of the spatial lightmanipulation means.
 12. A computer program product for calibrating aspectroscopic system, the spectroscopic system having a light source (1)for generating an excitation radiation (50) being adapted to betransmitted into a volume of interest (4), an objective (12) forcollecting return radiation from the volume of interest, a dispersiveoptical element (30) for spatially separating spectral components of thereturn radiation in a first direction and spatial light manipulationmeans (34) for modulating the spectral components of the returnradiation, the computer program product comprising computer programmeans being adapted to: (a) store a first output signal of an at leastfirst detector (40), the first output signal being generated in responseto detect a radiation being transmitted through a reference segment (36)of the spatial light manipulation means, (b) store a second outputsignal of the at least first detector after a modification of theposition of the spatial light manipulation means along the firstdirection and/or after a modification of the wavelength of theexcitation radiation and/or after a modification of the orientationand/or position of the dispersive optical element and/or after areconfiguration of a spatial transmission pattern of the spatial lightmanipulation means, (c) compare the first and second output signal ofthe at least first detector, (d) repeat steps (a) to (c) until theoutput signal is indicative of a maximum of radiation being detected bymeans of the at least first detector.